Perinatal exposures may lead to increased risk of childhood cancers, as well as those later in life. Preconceptional parental, transplacental, and/or neonatal exposures may be involved. Studies with animal models are utilized to increase understanding of underlying cellular and molecular mechanisms. We have been focusing on transgenerational effects of exposures and experiences of male mice on tumorigenesis and other endpoints in offspring. A possible mechanism of paternal effects on childhood cancer is male-mediated transgenerational carcinogenesis. Exposure of male mice to chromium(III), an environmental/occupational metal, results in increased neoplasms and other lesions in the offspring. The nature of these changes suggested hormonal involvement, and we have discovered alterations in serum glucose, corticosterone, IGF1, and the thyroid hormone T3 in the offspring. It is also important to discover the molecular mechanism in the sperm, by which changes-in-gene-expression signals are passed to offspring. Use of representational difference analysis, bisulfite sequencing, and pyrosequencing has revealed hypomethylation in the spacer-promoter region of the ribosomal RNA (rRNA) gene in sperm from Cr(III)-treated males. Ribosomal RNA is implicated in both growth and cancer. To test whether paternally-mediated effects are inherited by offspring, we have examined the gene in embryos and in tissues of young adult offspring, including liver and lung. Extensive statistical analyses of large numbers of litters and of offspring are now almost completed, and some surprising and interesing results are clear. Acidic saline, used as a vehicle for the Cr(III), also has had transgenerational effects. Importantly, offspring body weights and liver weights have been significantly increased as a result of the paternal treatments. The potential significance of this for several human diseases in addition to cancer is obvious. At the molecular level in studies of rRNA, single nucleotide polymorphisms, including one in the spacer-promoter and four alleles in the main promoter of the rRNA gene, are involved. The ratio of the alleles for the main promoter was actually changed by exposure of the fathers, selectively in the offspring lungs. This suggests tissue-specific genomic effects, either selective allelic expansion or gene conversion. If confirmed it will be a qualitatively new finding. The nature of the allele in the main promoter correlated with degree of methylation at five CpG sites in the spacer-promoter, in a tissue specific way. Degree of methylation was significantly affected by the paternal chromium treatment, in male offspring lungs. Importantly, these relationships between gene polymorphisms and gene methylation at a distant regulatory site were significantly modulated by treatment of the fathers. Since both the chromium(III), and the acid saline vehicle administered as a control, had clear effects, we looked for general, stressor-type effects in the males. Both chemical treatments were found to be stressors of the fathers, causing changes in serum corticosterone, insulin, leptin and glucose. Currently we are finding that these types of molecular changes also carry over into the F2 offspring, fathered by F1 males, that is, the grandchildren of the exposed males. Changes in the rRNA gene, both methylation and gene variant frequency, are greater than in the F1 mice This is particularly interesting in view of human epidemiology showing that experiences of grandfathers, for example amount of food available to them during certain intervals of childhood, affects risk of disease in their grandsons. To confirm these findings with another model and to simplify the experimental paradigm, we explored ways of exposing male mice directly to the stress hormone corticosterone. Preliminary trials have indicated that a key parameter is the age of these males at weaning, with different results obtained if the mice are weaned at 14 vs 21 days. This observation points to perinatal stress having a lasting effect on adult responses, a phenomenon worth study in its own right. Most consistent results were obtained with male weaned at 21 days. With such males, corticosterone treatment has an apparent effect on baseline weight and weight gain in the offspring. Statistical analysis of this result is in progress